This is an exploratory/development grant application to explore the role of apoptosis in corneal transplant failure. In spite of the use of immune suppression, many corneal grafts do not survive, and data suggest that graft endothelial cells are progressively lost for a long time after corneal transplantation. Our preliminary data provide support for the feasibility of introducing genes into both the corneal endothelium and stroma. Additionally, we have data suggesting that corneal transplants from transgenics that overexpress the anti-apoptotic factor Bcl-xL have a significantly enhanced survival rate, leading to our overarching hypothesis that downmodulation of apoptosis in the graft can promote graft survival. Based on this hypothesis, we have identified two aims: Specific Aim 1. Test the effect of overexpressing anti-apoptotic genes on a) the survival of cultured corneal endothelial cells (CEC) in vitro and b) survival of corneal grafts in vivo. For the in vitro studies, we will overexpress anti-apoptotic genes of interest (Bcl-2, Bcl-xL, and p35) and test the effect of such overexpression on survival of CEC following exposure to different types of apoptotic stimuli (H202, cytokines, and DNA damaging agents). The in vivo studies will complement the in vitro studies by testing the effect of select overexpression of these factors in transgenics on graft survival in a standard validated model of orthotopic corneal transplantation. Data from Specific Aim 1 will help identify the optimal gene(s) relevant for promoting corneal graft survival. Specific Aim 2. Test whether gene therapy with antiapoptosis genes reduces failure of corneal grafts in mice. Taken together, these aims will provide significant information on 1) overall role of graft cell apoptosis in affecting transplant survival, 2) differential effect of several potent anti-apoptotic factors on survival of corneal endothelial cells, and 3) feasible anti-apoptotic gene therapy interventions that can promote expression of desirable genes into corneal tissues. The long-term objective of this project is to identify anti-apoptotic strategies that may eventually be adopted for use in corneal transplantation, the most common form of tissue grafting, with nearly 40,000 cases performed annually in the United States alone. We believe that our goals are novel, innovative, and highly suited to the skills of our laboratory, and in delineating the role of apoptosis in corneal graft survival.